Progressive cardiac dilatation and pump failure of unknown etiology has been termed “idiopathic” dilated cardiomyopathy (DCM) (Richardson 1996 Circulation, 93, 841-842). DCM represents one of the main causes of severe heart failure with an annual incidence of up to 100 patients and a prevalence of 300-400 patients per million (AHA report 2007). Mutations in genes encoding myocyte structural proteins (Morita 2005) and several cardiotoxins, including alcohol, anthracyclines, and, more recently, therapeutically used monoclonal antibodies (e.g., trastuzumab) account for about one third of DCM cases (Chien 2000, Fabrizio and Regan 1994). The etiology of the remaining two thirds is still poorly understood, however.
At present the large majority of DCM is thought to arise from an initial (mostly viral) infection leading to acute myocarditis which upon activation of the immune system may progress to (chronic) autoimmune myocarditis resulting in cardiac dilatation and severe congestive heart failure; the latter progression occurs particularly, when associated (a) with the development of autoantibodies against distinct myocyte sarcolemmal or membrane proteins which are essential for cardiac function (Freedman 2004, Jahns 2006), or (b) with chronic inflammation of the myocardium and viral persistence (Kühl 2005). These recent findings are further strengthened by the fact, that patients with DCM often have alterations in both cellular and humoral immunity (Jahns 2006, Limas 1997, Luppi 1998, Mahrholdt 2006). Under such conditions an initial acute inflammatory reaction may proceed into a kind of low-grade inflammation (MacLellan 2003) facilitating the development of abnormal or misled immune responses to the primary infectious trigger (Freedman 2004, Kühl 2005, MacLellan and Lusis 2003, Maekawa 2007, Smulski 2006).
In the context of their humoral response a substantial number of DCM patients have been found to develop cross-reacting antibodies and/or autoantibodies to various cardiac antigens, including mitochondrial proteins (e.g., adenine nucleotide translocator, lipoamide and pyruvate dehydrogenase (Pohlner 1997, Schultheiss 1985, Schultheiss 1988, Schulze 1999)), sarcolemmal proteins (e.g., actin, laminin, myosin, troponin (Caforio 2002, Göser 2006, Li 2006, Neumann 1990, Okazaki 2003)), and membrane proteins (e.g., cell surface adrenergic or muscarinergic receptors (Christ. 2006, Fu 1993, Jahns 1999b, Magnusson 1994). From these, only a few selected antibodies appear to be able to cause myocardial tissue injury and to induce severe congestive heart failure by itself, however. In addition, the individual genetic predisposition (including the respective human leucocyte antigen (HLA)- and the major histocompatibility complex (MHC)-phenotype (Limas 1996)) may also significantly contribute to the susceptibility to self-directed immune reactions and the phenotypic expression of the disease (Limas 2004, MacLellan 2003).
Homologies between myocyte surface molecules such as membrane receptors and viral or bacterial proteins have been proposed as a mechanism for the elaboration of endogenous cardiac autoantibodies by antigen mimicry (Hoebeke 1996, Mobini 2004). Chagas' heart disease, a slowly evolving inflammatory cardiomyopathy, is one of the most prominent examples for this mechanism (Elies 1996, Smulski 2006). The disease originates from an infection with the protozoon Trypanosoma cruzi; molecular mimicry between the ribosomal P2β-protein of T. cruzi and the N-terminal half of the second extracellular loop of the β1-adrenergic receptor (β1-AR) results in generation of cross-reacting antibodies in about 30% of the Chagas' patients (Ferrari 1995). Because receptor-autoantibodies from patients with DCM preferentially recognize the C-terminal half of the same loop (Wallukat 1995), it was speculated that these antibodies might originate from molecular mimicry between the β1-AR and a hitherto unidentified viral pathogen (Magnusson 1996). Another—probably more relevant—mechanism leading to the production of endogenous cardiac autoantibodies would be primary cardiac injury followed by (sudden or chronic) liberation of a “critical amount” of antigenic determinants from the myocyte membrane or cytoplasm, previously hidden to the immune system. Such injury most likely occurs upon acute infectious (myocarditis), toxic, or ischemic heart disease (myocardial infarction) resulting in myocyte apoptosis or necrosis (Caforio 2002, Rose 2001). Presentation of myocardial self-antigens to the immune system may then induce an autoimmune response, which in the worst case results in perpetuation of immune-mediated myocyte damage involving either cellular (e.g., T-cell), or humoral (e.g., B-cell) immune responses, or co-activation of both the innate and the adaptive immune system (Eriksson 2003, Rose 2001).
From a pathophysiological point of view, it seems reasonable to link the harmful (e.g., cardiomyopathy-inducing) potential of a heart-specific autoantibody to the accessibility and to the functional relevance of the corresponding target. Myocyte surface receptors are easily accessible to autoantibodies (Okazaki 2005). The two most promising candidates are the cardiac β1-AR (representing the predominant adrenocepter subtype in the heart) and the M2-muscarinic acetylcholine receptor; against both receptors autoanti-bodies have been detected in DCM patients (Fu. 1993, Jahns 1999b, Matsui 1995). Whereas anti-muscarinic antibodies (exhibiting an agonist-like action on the cardiac M2 acetylcholine-receptor) have been mainly associated with negative chronotropic effects at the sinuatrial level (e.g., sinus node dysfunction, atrial fibrillation (Baba 2004, Wang. 1996)), agonistic anti-β1-AR antibodies have been associated with both the occurrence of severe arrhythmia at the ventricular level (Christ 2001, Iwata 2001a), and the development of (maladaptive) left ventricular hypertrophy, finally switching to left ventricular enlargement and progressive heart failure (Iwata 2001b, Jahns 1999b, Khoynezhad 2007). Both autoantibodies appear to be directed against the second extracellular loop of the respective receptors. To generate an autoimmune response, myocyte membrane proteins (e.g., receptors) must be degraded to small oligopeptides able to form a complex with a MHC or HLA class II molecule of the host (Hoebeke 1996). In case of the human β1-AR computer-based analysis for potential immunogenic amino-acid stretches has shown, that the only portion of the receptor molecule containing B- and T-cell epitopes and being accessible to antibodies was in fact the predicted second extracellular receptor loop (β1-ECII) (Hoebeke 1996). This might explain the successful use of second loop-peptides for the generation of β1-specific receptor antibodies in different animal-models (Iwata 2001b, Jahns. 2000, Jahns 1996). Moreover, in the last decade several groups have independently demonstrated that second loop antibodies preferentially recognize intact native β1-AR in various immunological assays (whole cell-ELISA, immunoprecipitation, immunofluorescence), indicating that they are “conformational” (Hoebeke 1996, Jahns 2006). Functional testing revealed that the same antibodies also affected receptor function, such as intracellular cAMP-production and/or cAMP-dependent protein kinase (PKA) activity, suggesting that they may act as allosteric regulators of β1-AR activity (Jahns 2000, Jahns 2006). The structure of the β1-AR was also analyzed by Warne (2008 Nature. DOI:10. 1038).
Following Witebsky's postulates (Witebsky 1957) indirect evidence for the autoimmune etiology of a disease requires identification of the trigger (e.g., the responsible self-antigen), and induction of a self antigen-directed immune response in an experimental animal, which then must develop a similar disease. Direct evidence, however, requires reproduction of the disease by transfer of homologous pathogenic antibodies or autoreactive T-cells from one to another animal of the same species (Rose 1993).
To analyze the pathogenetic potential of anti-β1-AR antibodies, Jahns et al. has chosen an experimental in vivo approach, which met the Witebsky criteria for direct evidence of autoimmune diseases. DCM was induced by immunizing inbred rats against β1-ECII (100% sequence homology between human and rat; indirect evidence); then the disease was reproduced in healthy animals by isogenic transfer of rat anti-β1-AR “autoantibodies” (direct evidence) (Jahns 2004). The animals developed progressive left ventricular (LV)-dilatation and dysfunction, a relative decrease in LV wall-thickness, and selective downregulation of β1-AR, a feature that is also seen in human DCM (Lohse 2003).
These results, together with an agonist-like short-term effect of the antibodies in vivo (Jahns 2004), suggest that both the induced and the transferred cardiomyopathic phenotypes can be attributed mainly to the mild but sustained receptor activation achieved by stimulatory anti-β1-AR antibodies. This hypothesis is supported by the large body of data available on the cardiotoxic effects of excessive and/or long term β1-AR activation seen after genetic or pharmacological manipulation (Engelhardt 1999, Woodiwiss 2001). Therefore, anti-β1-AR induced dilated immune-cardiomyopathy (DiCM) can now be regarded as a pathogenetic disease entity of its own, together with other established receptor-directed autoimmune diseases such as myasthenia gravis or Graves' disease (Freedman 2004, Hershko 2005, Jahns 2004, Jahns 2006).
The clinical importance of cardiac autoantibodies is difficult to assess, since low titers of such antibodies can also be detected in the healthy population as a part of the natural immunologic repertoire (Rose 2001). However, regarding functionally active anti-β1-AR antibodies previous data from Jahns et al. has demonstrated that their prevalence is almost negligible in healthy individuals (<1%) provided that a screening procedure based on cell-systems presenting the target (e.g., the β1-AR) in its natural conformation is used (Jahns 1999b). By employing the latter screening method, occurrence of anti-β1-AR autoantibodies could also be excluded in patients with chronic valvular or hypertensive heart disease (Jahns 1999a). In contrast, the prevalence of stimulating anti-β1-AR was ˜10% in ischemic (ICM) and ˜30% in dilated cardiomyopathy (DCM) (Jahns 1999b), which was significantly higher than in healthy controls, but in the lower range of previous reports on DCM collectives (33% to 95% prevalence) (Limas 1992, Magnusson 1994, Wallukat 1995). It seems conceivable that differences in screening methods aiming to detect functionally active anti-β1-AR autoantibodies most likely account for the wide range of prevalences reported in the past (Limas 1992). In fact, only a minor fraction of ELISA-defined human anti-β-AR autoantibodies was able to bind to cell surface located native β-AR. Only this fraction recognized (as determined by immunofluorescence) and activated (as determined by increases in cellular cAMP and/or PKA activity) human β1-AR expressed in the membrane of intact eukaryotic cells (Jahns 2000, Jahns 1999b). Therefore, cell systems presenting the target in its natural conformation represent an essential tool in the screening for functionally relevant anti-β-AR autoantibodies (Nikolaev 2007).
Clinically, the presence of anti-β1-AR autoantibodies in DCM has been shown to be associated with a more severely depressed cardiac function (Jahns 1999b), the occurrence of more severe ventricular arrhythmia (Chiale 2001), and a higher incidence of sudden cardiac death (Iwata 2001a). Recent data comparing antibody-positive with antibody-negative DCM patients over a follow-up period of more than 10 years not only confirmed a higher prevalence of ventricular arrhythmia in the presence of activating anti-β1-AR, but also revealed that antibody-positivity predicted an almost three-fold increased cardiovascular mortality-risk (Stork 2006). Taken together, the available clinical data underscore the pathophysiological relevance of functionally active anti-β1-AR antibodies in DCM.
One today generally accepted pharmacological strategy would be the use of beta-blocking agents in order to attenuate or even abolish the autoantibody-mediated stimulatory effects, at least if β-blockers can indeed prevent the antibody-induced activation of β1-AR (Freedman 2004, Jahns 2000, Matsui 2001, Jahns 2006). New therapeutic approaches actually include elimination of stimulatory anti-β1-AR by non-selective or selective immunoadsorption (Hershko 2005, Wallukat 2002), or direct targeting of the anti-β1-ECII antibodies and/or the anti-β1-ECII producing B-cells themselves (that is, induction of immune tolerance) (Anderton 2001). Non-selective immunoadsorption, however, because of an increased risk of infection after immunoglobulin depletion, requires the substitution of human IgG on the ground of safety (Felix 2000) with all possible side effects of substituted human proteins known in the art including severe anaphylactic reactions and death.
WO 01/21660 discloses certain peptides homologous to epitopes of the 1st and the 2nd loop of β1-AR, and proposes to apply these peptides for medical intervention of dilatative cardiomyopathy (DCM). Even if WO 01/21660 mentions marginally that peptides may be modified in order to protect them against serum proteases, for example by cyclization, corresponding examples and embodiments are not given and any in vitro or in vivo effect of the proposed peptides on the course of DCM or on the course of receptor-antibody titers is not shown. Moreover, in WO 01/21660 intends to rely on the above mentioned non-selective immunoadsorption approaches bearing the correspondingly mentioned risks.
In contrast thereto, the newly developed β1-ECII-homologous cyclopeptides (e.g. β1-ECII-CPs) were employed six weeks after the active induction of stimulatory anti-β1-ECII antibodies. β1-ECII-CPs are cyclopeptides containing 3 cysteine residues and hence, can form intramolecular bonds, whereby there is a potential option to form two intramolecular bonds (besides the cyclization between the N- and C-terminus), individually. β1-ECII-CP significantly reduced the amount of circulating anti-β1-ECII antibodies and effectively prevented development of cardiac dilatation and dysfunction (Boivin 2005). The above-mentioned β1-ECII-CPs were also disclosed in WO 2006/103101.